Research and development have been conducted on so-called biosensors and bioreactors that utilize the molecular recognition function and/or substance conversion capability of biomaterials such as nucleic acid (DNA or RNA), enzymes and antibodies, aiming at a wide range of applications thereof.
Concerning the biosensor, further technological development is required to be applied for detection of a wide variety of objects, with increasing concern about problems of environmental pollutants, social safety and health. Concerning the bioreactor, it attracts attention more and more as an environmentally friendly clean process, and further technological development is required, for example development of production methods utilizing various bioprocesses.
More specifically, concerning the biosensor, detectors utilizing specificity of molecular recognition of a biomaterial are now widely developed. For example, there have been developed biosensors such as a DNA sensor chip utilizing base sequence-dependent complementary hydrogen bonding between deoxyribonucleic acid (DNA) strands (hybridization reaction between complementary strands); an antibody sensor for detecting disease markers and the like in the blood utilizing the molecular recognition function of an antigen-antibody reaction, that is, specific binding between a protein molecule and a small molecule or between protein molecules; and an enzyme sensor that detects the concentration of a substance utilizing an enzyme such as oxidoreductase and hydrolase, represented by a glucose sensor for diabetes patients.
Currently, these biosensors are generally used in a form of the biomaterial-immobilizing substrate in which a biomaterial such as nucleic acid molecules (e.g., DNA), and proteins such as antibodies and enzymes is immobilized on the surface of a substrate or carrier.
One of the performances required for developing biosensors is “high sensitivity and miniaturization” represented by μ-TAS (micro-Total Analysis Systems). For achieving the goal of “high sensitivity and miniaturization”, it is an important technical challenge how to increase the sensitivity as well as how to use the space of very small reaction or detection field effectively.
For example, in the detection field having a biomaterial immobilized on a substrate, non-specific adsorption of biomaterials in addition to the specific binding of the target substance may occur, or the target substance itself is nonspecifically adsorbed on the substrate. These nonspecific adsorption phenomena are one of the factors reducing the S/N ratio of the biosensor. Particularly, as the detection field decreases in size, the total amount of the specifically bound target substance decreases, making the influence of the noise due to nonspecific adsorption substantial, hampering highly sensitive measurement. Furthermore, in view of effective utilization of a sample in a very small amount, non-specific adsorption of the target substance makes measurement of sufficiently high accuracy difficult. Therefore, reduction and inhibition of nonspecific adsorption phenomena is an important technical challenge.
On the other hand, concerning the bioreactor, instead of direct use of microorganisms that can produce a desired product, food additives such as amino acids, pharmaceutical candidate substances and antibiotics are now produced by enzyme reactions utilizing the site-specific catalytic property of enzymes. Further, application of enzyme reaction in production of chemical products and polymer materials is now under development. In development of such bioreactors utilizing enzyme reactions, development of apparatuses suitable for small-quantity and multi-product production has become mainstream. For example, as the technique of screening of candidate substances by the combinatorial chemistry method has come into wide use, needs for downsizing of the apparatus for small quantity production are increasing, e.g., apparatuses having immobilized enzyme protein for reaction as with the case of the biosensor.
Furthermore, materials of the substrate or carrier for the biomaterial immobilization that are utilized in the biosensor and bioreactor are generally selected from known materials such as organic polymers, glass, ceramics and metal substrates depending on the type and application of the immobilized organic material or biomaterial.
When the target substance that specifically interacts with the immobilized organic material on the substrate is a biomaterial, especially a protein, and the substrate surface is hydrophobic, nonspecific adsorption of the target substance onto such a hydrophobic surface will increase. Thus, sufficient detection sensitivity cannot be achieved with the biosensor, and high productivity may not be achieved with the bioreactor.
One of methods for reducing nonspecific adsorption onto the hydrophobic surface is to render the substrate surface hydrophilic, at least part of the substrate surface such as channels and reaction fields that contact a liquid containing the target substance. From the substrate surface subjected to the hydrophilicity treatment, the target substance protein physically adsorbed on the surface can be removed relatively easily by washing with a cleaning aqueous solution of a desired composition. Popular methods for rendering the substrate surface hydrophilic include a method of providing on the surface a metal oxide layer represented by silicon oxide, and a method of forming a hydrophilic coat of a coupling agent represented by a silane coupling agent.
To immobilize biomaterials such as proteins on the surface of a substrate subjected to the hydrophilic treatment, there is, for example, immobilization of a protein on the substrate surface by physical adsorption by immersing the substrate in a protein solution or coating the substrate with a protein solution to form a coating layer of the protein solution on the substrate surface, and then removing/drying the solvent contained in the coating layer, or a method of chemical immobilization by chemically modifying the substrate surface or protein molecules for the purpose of introducing reactive functional groups, and then forming a chemical bond through a reaction between introduced reactive functional groups.
As one example of the physical adsorption immobilization method, Japanese Published Patent Application No. H06-003317 discloses a method for preparation of an enzyme electrode applying a method of forming an organic charge transfer complex layer on the surface of a conductive substrate, and then coating the organic charge transfer layer with a protein solution and then drying the layer to physically adsorb and immobilize an enzyme protein on the substrate surface via the organic charge transfer layer.
As one of the chemical immobilization method, Sensor and Actuators B 15-16 p 127 (1993) discloses a method of treating the platinum-deposited surface of a silicon substrate with an amine based silane coupling agent, and then using a cross-linking agent such as glutaraldehyde to link an amino group of the amino silane coupling with a peptide chain via chemical bonding. Another example is, in preparation of a detector such as a biosensor having an immobilized antibody on a glass substrate, a method that introduces reactive functional groups to the surface of a glass substrate by the silane coupling agent treatment, and similarly uses a cross-linking agent to immobilize peptide chains through chemical bonds.
However, in the method of utilizing chemical binding by the physical adsorption and cross-linking reaction to immobilize biomaterials, sites involved in adsorption on the protein side can not be freely selected in physical adsorption onto the substrate particularly when applying to proteins such as enzymes and antibodies. Furthermore, sites in which functional groups involved in the reaction exist on the protein side for the cross-linking agent cannot be freely set, and when a plurality of functional groups capable of reaction exist, selectivity among the groups is extremely low. That is, in binding to the substrate through chemical bonds by the physical adsorption and cross-linking reaction, sites involved in binding on the protein side are randomly selected, and therefore if the sites directly involved in or indirectly related to the capability of the protein binding to an object compound, the enzyme activity of the protein and the like are also involved in binding to the substrate surface, the capability of binding to the object and the enzyme activity, which are possessed by the protein, may be significantly reduced when the sites bond to the substrate.
Thus, development of means for previously setting immobilized sites on the non-immobilized molecule side, which are involved in binding to the substrate surface, for example a technique capable of previously controlling the orientation of biomaterials immobilized on the substrate surface becomes important.
In addition, in achievement of “high sensitivity and miniaturization”, it is necessary to highly integrate and immobilize biomaterials in very small areas on the substrate surface.
As one example of the method for highly integrating and immobilizing biomaterials, a method of employing as a substrate a substrate having a large specific surface area, for example a porous material having a regular nano-level microporous structure, and immobilizing biomaterials on the surface having the microporous structure having a large specific surface area is generally known. For the method of forming a regular microporous structure on the scale of nanometer order capable of being used for the above-described application, polymer-processed membrane filters, porous glass, anode-oxidized aluminum films, and the like are well known. Furthermore, a method of forming a porous coated film on the surface of a substrate such as a metal or glass by a coating process is also known. In the method described in Science 279 548 (1997), as a method for forming the porous film, a structure having silica formed around a nonionic block polymer as a mold under acidic conditions using alkoxy silane as a starting material is first formed. Then, the nonionic block polymer as a mold is eliminated by heating or treatment with an organic solvent, whereby a silica film of porous structure having a pore diameter in the order of several nanometers. By using as a substrate such a porous material having a pore diameter in the order of several nanometers, a reaction field having immobilized thereon biomaterials in an amount sufficient for high sensitive detection can be prepared even in a very small area.
Examples of the method utilizing the porous substrate, especially the method of immobilizing biomaterials such as proteins to a porous silicon oxide material may include methods described in the following documents.
Japanese Patent Application Laid-Open No. 2000-139459 discloses extremely stable enzymes such as peroxidase, subtilisin and lipase immobilized in micropores of a mesoporous silica porous material having an anionic surface by means of the van der Waals force.
Japanese Patent Application Laid-Open No. 2001-128672 also discloses a method of degrading a lignin substrate by peroxidase immobilized in micropores of a mesoporous silica porous material.
Further, also using a method of immobilizing an enzyme protein on a porous substrate, Japanese Patent Application Laid-Open No. 2001-46100 discloses a method for enzymically modifying a fuel, Japanese Patent Application Laid-Open No. 2001-178457 discloses a method for immobilizing an enzyme, and Japanese Patent Application Laid-Open No. 2002-95471 discloses a method for improving the substrate specificity of lipase, a lipid-degrading enzyme.
Further, it is reported that immobilization of enzyme proteins such as cytochrome c, papain and trypsin in a mesoporous molecular sieve of silicon oxide improves the performance of these enzymes (Journal of Molecular Catalysis. B, 2(2-3), 115-126 (1996)).
It is also reported that immobilization of an enzyme protein (α-chymotrypsin) in micropores of nanoporpus sol-gel glass by covalent binding utilizing a silanizing agent (trimethoxyl propanal) having an aldehyde group at the terminus can improve the stability of the enzyme protein (Biotechnology and Bioengineering, 74 (3), 249-255 (2000)).